A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Summary

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Abstract

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention.

Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation.

Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Introduction

The tumor microenvironment actively participates in most, if not all, aspects of tumorigenesis and cancer progression, including metastasis formation and the development of resistance to therapeutics¹. This stresses the need for preclinical orthotopic cancer mouse models that allow dissection of the complex tumor-stroma interactions occurring in the tumor niche.

Among the many cellular components of the tumor microenvironment, mesenchymal stem cells (MSCs) strongly contribute to cancer progression in multiple cancer types such as breast cancer, prostate cancer, brain tumors, multiple myeloma, and osteosarcoma

Protocol

Human adipose tissues for mesenchymal stem cell isolation were obtained from the department of Plastic Surgery of the Tergooi Hospital (Hilversum, Netherlands) after approval by the Institutional Ethical Committee and written informed consent. GFP-positive adipose MSCs were obtained from the Department of Medical and Surgical Sciences for Children and Adults (University of Modena and Reggio Emilia).

Animal experiments were performed in accordance with the Dutch law on animal experimentation, and the protocol was approved by the committee on animal experimentation of the VU University medical center, Amsterdam, Netherlands.

Results

In this study, we explored the ability of osteosarcoma-secreted EVs to educate MSCs towards a pro-tumorigenic and pro-metastatic phenotype. We show that osteosarcoma cells release exosome-like EVs that are internalized by MSCs. We measured the alteration of the MSC cytokine expression profile induced by cancer EVs, and evaluated the effect of the EV-educated MSCs on tumor growth and metastasis formation. The general representation of the study design is illustrated in

Discussion

Tumor-secreted extracellular vesicles (EVs) can alter the physiology of local and distant mesenchymal cells to generate a tumor-supportive environment. Here we describe the generation of a preclinical mouse model of osteosarcoma that allows dissection of the EV-mediated interactions between tumor cells and mesenchymal stem cells (MSCs) in vivo. We show that systemic injection of human tumor EV-educated MSCs in mice bearing osteosarcoma xenografts strongly promotes cancer growth and metastasis formation by activa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
Ultra Centrifuge	Beckman	Optima L-90K
Rotor SW32Ti	Beckman	369650	Referred to in the manuscript as ultra-swinging bucket rotor
Transmission electron microscope	Zeiss	EM109	Or similar TEM
Digital camera	Nikon	DMX 1200F	Or similar camera
Imaging software TEM	Nikon	ACT-1
Fluorescence microscope	Zeiss	Imager.D2	Or similar Fluorescence microscope
Imaging software FM	Zeiss	ZEN Blue
Incubator	Nuaire	4750E
Centrifuge	Hettick	ROTANTA 460R
-80 Freezer	Thermo electro corporation	n.a.
FACS	BD	BD FACScalibur	Or similar flow cytometer
Drill	Ferm	FCT-300	With 0.8 mm drill
HSS micro twist drills, 0.8 mm	Proxxon	28 852	0.8 mm drill
IVIS camera	Xenogen	Ivis Lumina	Referred to in the manuscript as bioluminescence camera. Xenogen is now part of Perkin Elmer
Living image software2.60	Xenogen / Igor Por	n.a	Xenogen is now part of Perkin Elmer
10 µL Syringe	Hamilton	Neuros Model 1701 RN
Needle: Hamilton RN Needle for Syringe, 26 Gauge, Pointstyle AS, custom length 2 cm	Hamilton	n.a.
Caliper	Mitutoyo	G08004463
Autoclave	Astell	n.a.
Heat Lamp	Philips	n.a.
Culture media
Fetal Bovine Serum	Hyclone	RYG35912
Platelet Lysate	n.a.	n.a.
IMDM medium	Lonza	BE12-722F
alpha-MEM medium	Lonza	BE02-002F
DMEM medium	Lonza	BE12-614F
pen/strep/glutamine	GIBCO	10378-016
heparin	LEO	012866-08
Trypsin/EDTA (10x)	GIBCO	15400-054
Cells
adipose deriverd MSCs	n.a.	n.a.
GFP-positive MSCs	n.a.	n.a.
human fibroblasts	n.a.	n.a.
143B cells	ATCC	CRL-8303
FLUC-143B cells	ATCC	CRL-8303	Transduced
Disposables
Culture flasks 175 cm2	CELLSTAR	660175
50 mL tubes	Greiner bio-one	210261
Freeze tubes	Thermoscientific	377224
Ultra-Clear tubes	Beckman	344058	Referred to in the manuscript as ultra-centrifuge tubes
0,22 µm filter	Millex	SLGV033RS
200 mesh Formvar-carbon-coated nickel grids	EMS (Electron Microscopy Sciences)
0.5 mL insulin syringes with 29G Needle	Terumo	U-100
Petri dish	Sigma - Aldrich	P7612
Filter paper	Thermo fisher Scientific	50363215
Reagents / kits
paraformaldehyde	Alfa Aeser	43368.9M
PBS	Braun	220/12257974/110
glutaraldehyde	EMS (Electron Microscopy Sciences)	16300
uranyl oxalate	EMS (Electron Microscopy Sciences)	22510
urany acetate	EMS (Electron Microscopy Sciences)	22400
methyl cellulose	EMS (Electron Microscopy Sciences)	1560
PKH67	Sigma	mini67-1kt	Referred to in the manuscript as GFLD
BSA	Sigma	A8412
CBA - human inflammatory cytokine kit	BD	551811
Formaldehyde 37%	VWR	104003100
Carbon Steel surgical blades	Swann-Morton	206	Referred to in the manuscript as surgical knife
anti-human vimentin antibody	Santa Cruz	sc-6260	Clone V9
Antibody diluent	DAKO	S0809
HRP-labeled anti mouse IgG antibody	Life Technologies	32230
DAB-kit	DAKO	K500711
hematoxyllin	Sigma	GHS232
EDTA-buffer	n.a.	n.a.
Citrate buffer	n.a.	n.a.
rabbit polyclonal anti-GFP antibody	Abcam	n.a.	Ab290
DAPI	Life Technologies	D1306
Paracetamol, 120 mg / 5 ml syrup	Bayer	n.a.	Sinaspril, paracetamol solution for kids
Isoflurane 1000 mg/g	Vumc pharmacy	n.a.
buprenofine hydrochloride, 0.3 mg/ml	Indivior UK Limited	n.a.
lidocaine-HCL 2%	Vumc pharmacy	n.a.
70% ethanol	VWR	93003.1006
Tissue glue	Derma+Flex, formulated medical cyanoacrylate	Vygon	LB604060
Eyedrops: Vidisec Carbogel, 2 mg/ml	Bausch+Lomb	n.a.
D-luciferin, potassium salt	Gold Biotechnology	LUCK-1
Glass slides	Thermo scientific	630-0954
Stainless steel loops	n.a.	n.a.
Mice experiments
Mice, Hsd:Athymic Nude-Foxn1nu,  female, 6 weeks at arrival, bacterial status conform FELASA	ENVIGO	n.a.
Paper-pulp smart home (cage enrichment)	Bio Services	n.a.
Alpha-dri bedding material	Shepperd Speciality Papers	n.a.
Mouse food: Teklad global 18% protein rodent diet	ENVIGO	2918-11416M
Sutures	Ethicon	V926H
Scissors	Sigma-Aldrich	S3146-1EA	(or similar)
Tweezers	Sigma-Aldrich	F4142-1EA	(or similar)